RESUMO
The gastrointestinal peptide, peptide YY3-36 (PYY3-36) and its shorter peptide analogues have been reported to reduce appetite by activating the neuropeptide Y2 receptor (Y2R), which is associated with obesity and other metabolic diseases. A 14-amino acid PYY analogue, Ac-[d-Pro24,Cha27,28,36,Aib31]PYY(23-36) (3), showed high binding affinity and agonist activity for the Y2R, similar to that of PYY3-36, but had weak anorectic activity upon continuous administration in lean mice. Three amino acid substitutions [Pya(4)26, Aib28, Lys30], which contributed to the decreased hydrophobicity of 3, efficiently increased its anorectic activity. The compound containing these three amino acids, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (22), exerted more potent and durable food intake suppression than that by PYY3-36 in lean mice, as well as excellent Y2R agonist activity (EC50: 0.20nM) and good subcutaneous bioavailability (66.6%). The 11-day continuous administration of 22 at 1mg/kg/day successfully produced antiobese and antidiabetic effects, with more than 20% body weight loss in obese and Type 2 diabetes ob/ob model mice.
Assuntos
Fármacos Antiobesidade/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Obesidade/tratamento farmacológico , Peptídeo YY/farmacologia , Animais , Fármacos Antiobesidade/administração & dosagem , Fármacos Antiobesidade/química , Relação Dose-Resposta a Droga , Injeções Intravenosas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Estrutura Molecular , Peptídeo YY/administração & dosagem , Peptídeo YY/química , Receptores de Neuropeptídeo Y/agonistas , Relação Estrutura-AtividadeRESUMO
Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.
Assuntos
Fármacos Antiobesidade/farmacologia , Neuropeptídeos/farmacologia , Polietilenoglicóis/química , Animais , Fármacos Antiobesidade/farmacocinética , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/química , Neuropeptídeos/farmacocinética , Suínos , Redução de Peso/efeitos dos fármacosRESUMO
Continuous administration of a 14-amino acid peptide YY (PYY) analog, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (4), which has a high binding affinity and agonist activity for the neuropeptide Y2 receptor (Y2R), has previously shown an antiobesity effect in a 2-week diet-induced obesity (DIO) study in mice. However, there remained a possibility to obtain more potent analogs by further improving its pharmacokinetic profile. A combination of the N-terminal 4-imidazolecarbonyl moiety and three amino acid substitutions, trans-4-hydroxy-d-proline (d-Hyp)24, isovaline (Iva)25, and γ-methylleucine (γMeLeu)28, not only improved the binding affinity of the peptide for Y2R but also increased its anorectic activity in lean mice. In a 2-week DIO study in mice, continuous administration of 4-imidazolecarbonyl-[d-Hyp24,Iva25,Pya(4)26,Cha27,36,γMeLeu28,Lys30,Aib31]PYY(23-36) (31, PYY-1119) at a dose of 0.03mg/kg/day showed a highly potent antiobesity effect, with more than 10% body weight reduction.
Assuntos
Peso Corporal/efeitos dos fármacos , Peptídeo YY/química , Peptídeo YY/farmacologia , Sequência de Aminoácidos , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Dieta , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Knockout , Estrutura Molecular , Peptídeo YY/agonistasRESUMO
Neuromedin U (NMU) is a neuropeptide found in the brain and gastrointestinal tract. The NMU system has been shown to regulate energy homeostasis by both a central and a peripheral mechanism. Peripheral administration of human NMU-25 was recently shown to inhibit food intake in mice. We examined the possibility that other NMU-related peptides exert an anorectic activity by intraperitoneal (i.p.) administration. We found that rat NMU-23 and its structurally-related peptide rat neuromedin S (NMS) significantly reduced food intake in lean mice, whereas NMU-8, an active fragment of the octapeptide sequence conserved in porcine, human and mouse NMU, had no effect. When rat NMU-23, NMU-8, and rat NMS were covalently conjugated to polyethylene glycol (PEG) (PEGylation) at the N-terminus of these peptides, PEGylated NMU-8 showed the most long-lasting and robust anorectic activity. The exploration of the linker between NMU-8 and PEG using hetero-bifunctional chemical cross-linkers led to an identification of PEGylated NMU-8 analogs with higher affinity for NMU receptors and with more potent anorectic activity in lean mice. The PEGylated NMU-8 showed potent and robust anorectic activity and anti-obesity effect in diet-induced obesity (DIO) mice by once-daily subcutaneous (s.c.) administration. These results suggest that PEGylated NMU-8 has the therapeutic potential for treatment of obesity.
Assuntos
Depressores do Apetite/farmacologia , Neuropeptídeos/farmacologia , Obesidade/tratamento farmacológico , Animais , Depressores do Apetite/administração & dosagem , Ingestão de Alimentos/efeitos dos fármacos , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/administração & dosagem , Neuropeptídeos/química , Polietilenoglicóis/química , RatosRESUMO
Gastrointestinal peptides such as peptide YY (PYY) can regulate appetite, which is relevant to the study of obesity. The intraperitoneal bolus administration of PYY3-36 and a 12-amino acid PYY analogue, benzoyl-[Cha27,28,36,Aib31]PYY25-36 (1), showed similar anorectic activity by activating the Y2 receptor (Y2R). However, food intake inhibition and body weight loss were not observed upon continuous subcutaneous administration of 1 with osmotic pumps in diet-induced obese (DIO) mice. N-Terminal elongation of 1, together with amino acid substitution at position 24, led to a hydrophilic 14-amino acid peptide, Ac-[d-Hyp24,Cha27,28,36,Aib31]PYY23-36 (18), that showed higher affinity and more potent agonist activity for Y2R and a robust anorectic activity with potency similar to that of PYY3-36. In addition, the continuous subcutaneous administration of 18 at 0.3 mg/(kg·day) induced significant body weight loss in DIO mice. These results suggest that a short-length PYY analogue can be a lead compound for antiobesity therapy in a sustained-release formulation.
RESUMO
Peripheral administration of PYY3-36, a fragment of peptide YY (PYY), has been reported to reduce food intake by activating the neuropeptide Y2 receptor (Y2R). An N-terminally truncated PYY analogue, benzoyl-[Ala26,Ile28,31]PYY(25-36) (1), showed a relatively potent agonist activity for Y2R but a weak anorectic activity by intraperitoneal administration (2000 nmol/kg) in lean mice because of its markedly poor biological stability in the mouse serum. Notably, two cyclohexylalanine (Cha) substitutions for Tyr residues at positions 27 and 36 (4) improved the stability in the mouse serum concomitant with enhanced anorectic activity. Further optimization at positions 27, 28, 30, and 31 revealed that 21, containing Cha28 and Aib31 residues, showed a more potent anorectic activity than PYY3-36 at a low dose of 300 nmol/kg. The minimum effective dose by intraperitoneal administration of 21 was 30 nmol/kg (ca. 52 µg/kg) in mice, suggesting the biologic potential of short-length PYY3-36 analogues with a potent anorectic effect.
RESUMO
Metastin/kisspeptin is an endogenous ligand of KISS1 Receptor (KISS1R). Metastin and KISS1R are suggested to play crucial roles in regulating the secretion of gonadotropin-releasing hormone (GnRH), and continuous administration of metastin derivatives attenuated the plasma testosterone levels in male rats. Our optimization studies of metastin derivatives led to the discovery of 1 (Ac-d-Tyr-d-Trp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2, TAK-683), which suppressed plasma testosterone in rats at lower doses than those of leuprolide. Although 1 possessed extremely potent pharmacological activity, 20 mg/mL aqueous solution of 1 has a gel formation property. In order to improve this physicochemical property, we substituted d-Trp at position 47 with a variety of amino acids; we identified that substitution with cyclic amino acids, which could change peptide conformation, retained its potency. Especially, analogue 24 (TAK-448) with trans-4-hydroxyproline (Hyp) at position 47 showed not only superior pharmacological activity to 1 but also excellent water solubility. Furthermore, 20 mg/mL aqueous solution of 24 did not show gel formation up to 5 days.
Assuntos
Kisspeptinas/química , Kisspeptinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Testosterona/antagonistas & inibidores , Animais , Células CHO , Cricetulus , Humanos , Kisspeptinas/administração & dosagem , Kisspeptinas/sangue , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Solubilidade , Testosterona/sangue , Testosterona/metabolismoRESUMO
Metastin is encoded by a putative human metastasis suppressor gene KiSS-1, and is the cognate ligand of a G-protein-coupled receptor designated OT7T175. To study the physiological function(s) of metastin, we cloned rat and mouse KiSS-1 cDNAs both encoding 130-amino acid KiSS-1 proteins. Sequence analysis suggested that processing of the rat and mouse KiSS-1 proteins produces 52-amino-acid peptides, each with an amidated carboxyl terminal and with a single possible disulfide bond, corresponding to rat and mouse metastins. The carboxyl-terminal sequence of metastin, known to be essential for functional receptor interaction, was found to be highly conserved among humans and rodents. Real-time PCR analysis indicated that rat KiSS-1 mRNA showed the highest expression level in the cecum and colon. Since KiSS-1 mRNA and metastin are known to be abundant in human placenta, we further studied the localization of KiSS-1 and OT7T175 mRNAs in rat placenta by in situ hybridization. KiSS-1 and OT7T175 mRNAs were specifically detected in trophoblast giant cells at embryonic day 12.5, and the transcripts in the cells gradually decreased during placental maturation. These results suggest that metastin/OT7T175 signaling may participate in implantation of the mammalian embryo, placenta formation, and maintenance of pregnancy.
Assuntos
Genes Supressores de Tumor , Células Gigantes/metabolismo , Placenta/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar/metabolismo , Dissulfetos , Relação Dose-Resposta a Droga , Implantação do Embrião , Embrião de Mamíferos/metabolismo , Hibridização In Situ , Kisspeptinas , Camundongos , Dados de Sequência Molecular , Metástase Neoplásica , Peptídeos/química , Proteínas/genética , Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Fatores de Tempo , Distribuição Tecidual , Trofoblastos/metabolismoRESUMO
Galanin-like peptide (GALP) is a novel peptide that has been isolated from the porcine hypothalamus. The expression of GALP mRNA is localized to the hypothalamic arcuate nucleus and is thought to be under the regulation of leptin. First, we confirmed by real-time PCR analysis that sc administration of leptin to Wistar rats under food-deprived conditions resulted in a 1.5-fold increase in hypothalamic GALP mRNA levels. Next, GALP mRNA levels were found to be reduced by 50% in 11-wk-old male Zucker obese rats compared with age-matched Zucker lean rats, whereas neuropeptide Y mRNA levels were increased by 55% and proopiomelanocortin mRNA levels were reduced by 53% in Zucker obese rats. Analysis using a two-site enzyme immunoassay revealed a lower level of hypothalamic GALP immunoreactivity in 11-wk-old Zucker obese rats (5.9 fmol/mg protein) than in age-matched Zucker lean rats (19.6 fmol/mg protein). Immunohistochemical studies demonstrated that Zucker obese rats (11 wk old) had a reduced number of GALP immunoreactivity-positive cells (29.4 cells/3 slices) in the arcuate nucleus compared with age-matched Zucker lean rats (115 cells/3 slices). Furthermore, Zucker obese rats showed increased sensitivity to intracerebroventricularly administered GALP compared with Zucker lean rats, in that a lower dose of GALP increased plasma LH levels in male Zucker obese rats, but not in male Zucker lean rats. In addition, a reduction in the level of hypothalamic GALP mRNA was found in db/db and ob/ob mice. The result supports the hypothesis that the hypothalamic GALP gene expression is controlled by leptin signals and suggests possible involvement of GALP in the reproductive abnormalities of the Zucker obese rat.
Assuntos
Núcleo Arqueado do Hipotálamo/fisiologia , Leptina/metabolismo , Proteínas do Tecido Nervoso/genética , Obesidade/fisiopatologia , Animais , Núcleo Arqueado do Hipotálamo/química , Núcleo Arqueado do Hipotálamo/citologia , Modelos Animais de Doenças , Peptídeo Semelhante a Galanina , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Injeções Intraventriculares , Leptina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/farmacologia , Neurônios/química , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ratos Zucker , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
Kisspeptin/metastin, a hypothalamic peptide, plays a pivotal role in controlling gonadotropin-releasing hormone (GnRH) neurons, and we have shown that continuous subcutaneous administration of kisspeptin analogues suppresses plasma testosterone in male rats. This study examined pharmacologic profiles of investigational kisspeptin analogues, TAK-448 and TAK-683, in male rats. Both analogues showed high receptor-binding affinity and potent and full agonistic activity for rat KISS1R, which were comparable to natural peptide Kp-10. A daily subcutaneous injection of TAK-448 and TAK-683 (0.008-8µmol/kg) for consecutive 7 days initially induced an increase in plasma luteinizing hormone and testosterone levels; however, after day 7, plasma hormone levels and genital organ weights were reduced. Continuous subcutaneous administrations of TAK-448 (≥10pmol/h, ca. 0.7nmol/kg/day) and TAK-683 (≥30pmol/h, ca. 2.1nmol/kg/day) induced a transient increase in plasma testosterone, followed by abrupt reduction of plasma testosterone to castrate levels within 3-7 days. This profound testosterone-lowering effect was sustained throughout 4-week dosing periods. At those dose levels, the weights of the prostate and seminal vesicles were reduced to castrate levels. These suppressive effects of kisspeptin analogues were more rapid and profound than those induced by the GnRH agonist analogue leuprolide treatment. In addition, TAK-683 reduced plasma prostate specific antigen (PSA) in the JDCaP androgen-dependent prostate cancer rat model. Thus, chronic administration of kisspeptin analogues may hold promise as a novel therapeutic approach for suppressing reproductive functions and hormone-related diseases such as prostate cancer. Further studies are warranted to elucidate clinical significance of TAK-448 and TAK-683.
Assuntos
Antineoplásicos/farmacologia , Kisspeptinas/farmacologia , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Células CHO , Cálcio/metabolismo , Cricetulus , Kisspeptinas/sangue , Kisspeptinas/farmacocinética , Kisspeptinas/uso terapêutico , Leuprolida/farmacologia , Leuprolida/uso terapêutico , Hormônio Luteinizante/sangue , Masculino , Tamanho do Órgão/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/crescimento & desenvolvimento , Neoplasias da Próstata/tratamento farmacológico , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Glândulas Seminais/efeitos dos fármacos , Glândulas Seminais/crescimento & desenvolvimento , Testículo/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , Testosterona/sangueRESUMO
Metastin/kisspeptin, a hypothalamic peptide, plays a pivotal role in controlling GnRH neurons. Here we studied the effect of chronic sc administration of two kisspeptin analogs, KISS1-305 and TAK-448, on hypothalamic-pituitary-gonadal function in male rats in comparison with a GnRH analogue leuprolide or bilateral orchiectomy (ORX). The prototype polypeptide, KISS1-305 (1-4 nmol/h), caused substantial elevations of plasma LH and testosterone, followed by abrupt reductions of both hormone levels. Notably, testosterone levels were reduced to castrate levels within 3 d and remained depleted throughout the 4-wk dosing period, an effect that was faster and more pronounced than leuprolide (1 nmol/h) dosing. KISS1-305 also reduced genital organ weight more profoundly than leuprolide. In mechanistic studies, chronic KISS1-305 administration only transiently induced c-Fos expression in GnRH neurons, suggesting that GnRH-neural response was attenuated over time. Hypothalamic GnRH content was reduced to 10-20% of control at 3 wk without any changes in Gnrh mRNA expression. Dosing with the investigational peptide TAK-448 was also studied to extend our understanding of hypothalamic-pituitary functions. Similar to ORX, TAK-448 (0.1 nmol/h) depleted testosterone and decreased GnRH content by 4 wk. However, in contrast to ORX, TAK-448 decreased gonadotropin levels in pituitary and plasma samples, implying the suppression of GnRH pulses. These results suggest that chronic administration of kisspeptin analogs disrupts endogenous kisspeptin signals to suppress intrinsic GnRH pulses, perhaps by attenuating GnRH-neural response and inducing continuous GnRH leakage from the hypothalamus. The potential utility of kisspeptin analogs as novel agents to treat hormone-related diseases, including prostate cancer, is discussed.
Assuntos
Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Kisspeptinas/farmacologia , Neurônios/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testosterona/sangue , Animais , Hormônio Liberador de Gonadotropina/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Leuprolida/farmacologia , Hormônio Luteinizante/sangue , Masculino , Neurônios/metabolismo , Orquiectomia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Ratos , Ratos Sprague-Dawley , Testículo/metabolismoRESUMO
Metastin is a novel peptide that has been isolated from the human placenta as the cognate ligand of the G-protein-coupled receptor OT7T175 (or GPR54). However, its physiological functions have not yet been fully investigated. In the present study, we show that subcutaneous administration of metastin increased the plasma levels of gonadotropins (follicle-stimulating hormone and luteinizing hormone) and induced ovulation in prepubertal female rats that had been pretreated with pregnant mare serum gonadotropin to induce follicle maturation. Furthermore, metastin administration drastically increased the plasma levels of gonadotropins in male rats. This action was abolished by pretreatment with a GnRH antagonist, and was accompanied by induction of c-Fos immunoreactivity in GnRH neurons. These results suggest that s.c. administered metastin induces the release of gonadotropin via activation of the hypothalamic GnRH neurons.
Assuntos
Gonadotropinas/metabolismo , Ovulação/efeitos dos fármacos , Animais , Masculino , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos WistarRESUMO
Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.